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Determination of clostebol residues in the urine of slaughter animals using liquid chromatography–tandem mass spectrometry

Iwona Matraszek-Żuchowska

Iwona Matraszek-Żuchowska

Department of Pharmacology and Toxicology, National Veterinary Research InstitutePuławy, Poland
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Matraszek-Żuchowska, Iwona
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Alicja Kłopot

Alicja Kłopot

Department of Pharmacology and Toxicology, National Veterinary Research InstitutePuławy, Poland
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Kłopot, Alicja
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Justyna Grzelak

Justyna Grzelak

Department of Pharmacology and Toxicology, National Veterinary Research InstitutePuławy, Poland
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Grzelak, Justyna
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Paulina Zdonek

Paulina Zdonek

Department of Pharmacology and Toxicology, National Veterinary Research InstitutePuławy, Poland
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Zdonek, Paulina
  
31 dic 2024
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Pubblicato online: 31 dic 2024
Pagine: 611 - 621
Ricevuto: 19 lug 2024
Accettato: 20 dic 2024
DOI: https://doi.org/10.2478/jvetres-2024-0070
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© 2024 Iwona Matraszek-Żuchowska et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1.

Chemical structures of clostebol acetate and its metabolites
Chemical structures of clostebol acetate and its metabolites

Fig. 2.

Liquid chromatography–tandem mass spectrometry chromatograms of A – a blank animal urine sample (mixed bovine and porcine urine); B – a blank urine sample spiked with 17α/β-CLOS and CLAD at the individual CCα level; C – a blank urine sample spiked with 17α/β-CLOS and CLAD at MMPR=0.50 μg L−1; D – a compliant routine bovine urine sample; E – a compliant routine porcine urine sample; F – a compliant routine ovine urine sample; G – a compliant routine equine urine sample. Note: chromatograms show transitions used for quantification for each analyte according to Table 1
Liquid chromatography–tandem mass spectrometry chromatograms of A – a blank animal urine sample (mixed bovine and porcine urine); B – a blank urine sample spiked with 17α/β-CLOS and CLAD at the individual CCα level; C – a blank urine sample spiked with 17α/β-CLOS and CLAD at MMPR=0.50 μg L−1; D – a compliant routine bovine urine sample; E – a compliant routine porcine urine sample; F – a compliant routine ovine urine sample; G – a compliant routine equine urine sample. Note: chromatograms show transitions used for quantification for each analyte according to Table 1

LC-MS/MS ion acquisition parameters used for the identification of 17β-clostebol (17β-CLOS), 17α-clostebol (17α-CLOS) and 4-chloroandrostenedione (CLAD)

Compound MRM transition (m/z) Collision energy(V) Declustering potential(V) Entrance potential(V) Collision cell exit potential (V) ± standard deviation Samples fulfilling the confirmation criteria (%)
CCα – 2.50 μg L−1
17β-CLOS 323.2 > 143.1a 32 - -
323.2 > 131.0 33 70 10 24 0.532 ± 0.028 100.0
323.2 > 95.2 52 0.048 ± 0.009 99.1
17α-CLOS 323.3 > 143.0a 40 - -
323.3 > 131.0 31 70 11 20 0.381 ± 0.021 100.0
323.3 > 95.0 53 0.141 ± 0.018 97.2
CLAD 321.0 > 143.0a 31 - -
321.0 > 131.0 32 70 10 20 0.551 ± 0.015 100.0
321.0 > 95.2 53 0.056 ± 0.008 99.1
17β-CLOS-D3 326.4 > 143.0a 31 75 9 22 - - -

Validation parameters of the liquid chromatography–tandem mass spectrometry method for the determination of 17β-clostebol (17β-CLOS), 17α-clostebol (17α-CLOS) and 4-chloroandrostenedione (CLAD) in animal urine

Parameter Number of samples Spiking level (μg L−1) Compound
17β-CLOS 17α-CLOS CLAD
Mean concentration (μg L−1)/Apparent recovery (%) n = 21 0.25 0.24/95.1 0.24/96.8 0.25/100.8
0.50 0.54/107.4 0.52/103.2 0.53/106.4
0.75 0.77/102.9 0.75/100.5 0.78/104.4
n = 9 1.00 0.97/96.9 0.99/98.5 0.96/96.3
2.50 2.49/99.6 2.50/100.1 2.50/100.0
n = 10 CCα 0.31/112.4 0.27/94.5 0.32/99.1
Repeatability (sr, μg L−1/RSD, %) n = 21 0.25 0.01/4.2 0.01/5.3 0.02/8.0
0.50 0.03/6.4 0.03/5.8 0.02/4.3
0.75 0.06/7.3 0.04/5.4 0.05/6.1
n = 9 1.00 0.05/5.0 0.05/4.8 0.03/3.5
2.50 0.04/1.5 0.11/4.3 0.10/3.9
n = 10 CCα 0.03/9.1 0.03/10.0 0.03/10.3
Within-lab reproducibility (sR, μg L−1/RSD, %) n = 21 0.25 0.02/6.3 0.02/8.0 0.03/12.3
0.50 0.05/9.7 0.05/8.8 0.03/6.4
0.75 0.08/10.9 0.06/8.0 0.07/9.2
n = 9 1.00 0.07/7.6 0.07/7.2 0.05/5.2
2.50 0.06/2.3 0.16/6.5 0.15/5.9
Decision limit (CCα, μg L−1) 0.28 0.29 0.32
Expanded measurement uncertainty (U, k = 2, %) 0.25 13 16 24
0.50 18 17 13
0.75 21 16 17
1.00 15 15 11
2.50 9 13 12
Matrix effect (%) 14 14 19
Ruggedness A/a factors tA/a 0.0600 0.5250 1.8583
B/b factors tB/b 0.3650 0.0550 1.1417
C/c factors tC/c 0.3950 0.2400 0.3000
D/d factors tD/d 0.0850 0.0250 0.3833
E/e factors tE/e 0.3950 0.0200 0.1917
F/f factors tF/f 0.3200 0.2200 0.7750
G/g factors tG/g 0.1200 0.1600 0.5500
All factors SI/i 0.01 0.01 0.02
Standard calibration curve Slope ± sb 1.0014 ± 0.2316 0.8378 ± 0.3434 0.8479 ± 0.2464
y-intercept ± sa -0.0808 ± 0.0658 -0.1093 ± 0.0530 -0.0767 ± 0.0390
Correlation coefficient 0.9975 0.9935 0.9969
Standard error 0.1819 0.2462 0.1714
Matrix-matched calibration curve Slope ± sb 0.8586 ± 0.2401 0.7704 ± 1.2009 0.7997 ± 0.2704
y-intercept ± sa 0.0293 ± 0.0263 -0.0007 ± 0.0444 0.0131 ± 0.0208
Correlation coefficient 0.9992 0.9999 0.9997
Standard error 0.0324 0.0121 0.0188
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Argomenti della rivista:
Scienze biologiche, Biologia molecolare, Microbiologia e virologia, Scienze della vita, altro, Medicina, Medicina veterinaria
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